1. Field of the Invention
This invention relates to a method and reagents for detecting methadone in a test sample such as urine. In particular, the invention relates to a fluorescence polarization immunoassay procedure for determining the presence or amount of methadone in a sample, tracer compounds used as reagents in such procedure, and immunogen compounds used to raise antibodies for use as reagents in such procedure.
2. Description of Related Art
Methadone is a synthetic narcotic analgesic which has been used in maintenance programs as a treatment for morphine and heroin addiction. Methadone staves off the acute effects of withdrawal from heroin, and also reduces or eliminates the postaddiction syndrome of anxiety, depression, and craving. Excessive use of this drug, however, may lead to habituation or addiction.
The biological fluid used most frequently for detecting methadone is urine. Other biological samples, however, such as serum, plasma or tissue can be used as test samples. In the past, methadone has been detected by a number of techniques including thin-layer chromatography (TLC), gas chromatography (GC) and high performance liquid chromatography (HPLC). These methods generally involve complicated chemical extractions of the drugs from the test sample, procedures which require highly trained personnel and lengthy assay times.
Binding assays have provided a preferable alternative to chemical methods such as GC, TLC and HPLC. Binding assays for detecting antigens and antibodies depend upon the immunological reactivity which characterizes these substances. Generally, these assays are collectively termed immunoassays.
Immunoassay techniques take advantage of the mechanisms of the immune systems of higher organisms, wherein antibodies are produced in response to the presence of antigens which are foreign to the organisms. One or more antibodies are produced in response to, and are capable of reacting with, a particular antigen, thereby creating a highly specific reaction mechanism which can be used in vitro to determine the presence or concentration of that particular antigen in a biological sample.
Competitive binding assays for measuring analytes of interest are based on the competition between the analyte in the test sample and a labeled reagent (i.e., tracer) for a limited number of binding sites on a binding member (e.g., and antibody) that is specific for both the analyte and tracer. The concentration of analyte the sample determines the amount of tracer that will bind to the antibody. The amount of tracer-antibody complex formed can be quantitatively measured and is proportional to the quantity of analyte in the test sample.
Fluorescence polarization techniques provide a quantitative means for measuring the amount of tracer-antibody complex formed in a competitive binding immunoassay. These techniques are based on the principle that a fluorescently labeled tracer rotates rapidly when excited by linearly polarized light, and fluorescent light emitted by the rotating tracer becomes partially depolarized due to the rapid rotation. As a result, the tracer will emit fluorescence with a degree of polarization inversely related to the tracer's rate of rotation, i.e., the higher the rotation the lower the polarization of the emitted light (or the greater the depolarization of the emitted light). The speed of rotation and the amount of depolarization decrease when the tracer becomes bound to a heavier molecule, such as when it becomes bound to the comparatively heavier antibody molecule. If a fluorescent tracer-antibody complex is excited by linearly polarized light, more of the emitted light remains polarized because the fluorophore is constrained from rapidly rotating. When a "free" tracer (i.e., tracer that is not bound to an antibody) is excited by linearly polarized light, its rotation is much faster than that of the complex, and therefore, the emitted light is depolarized.
The fluorescence polarization immunoassay (FPIA) is a homogenous assay wherein the final polarization readings are taken from a solution in which bound tracer is not separated from free tracer. In heterogeneous immunoassay procedures, the bound tracer must be separated from the free tracer before a reading can be taken.
By using standard preparations for comparsion with a test sample containing an unknown level of the analyte, the FPIA provides a quantitative means for measuring the amount of tracer-antibody complex formed in a competitive binding assay. This procedure is currently being employed by Abbott Laboratories in its commercially available TDx.RTM. Therapeutic Drug Monitoring System and is described in U.S. Pat. No. 4,269,511 and U.S. Pat. No. 4,420,568.
As disclosed in the '511 and '568 patents, because the tracer must compete with the analyte for binding to the antibody in a FPIA, the tracer must possess a molecular structure sufficiently similar to the analyte to enable the tracer to be recognized by an antibody specific for the analyte. For this reason, the tracer is also referred to as a fluorescently labeled analyte-analog, a substantial portion of which has the same spatial and polar organization as the analyte to define one or more determinants capable of competing with the analyte for the binding sites on the antibody.
A need exists for providing an assay and reagents for performing an accurate and sensitive FPIA for the detection of the presence of methadone in a sample.